The general goal of the research proposed in this application continues to be the molecular characterization of the cardiac contractile system during development and in response to different physiological and pathological stimuli. This goal is based on the belief that in order to understand cardiac contractility and its modulation during development, in response to hormones and work overload, it is essential to have a clear understanding of the phenotype(s) of the cardiac contractile apparatus and the mechanisms involved in its regulation. To address these issues we will continue the characterization of the cardiac myosin heavy chain (MHC) and Alpha-tropomyosin (Alpha-TM) gene nucleotide sequences and will further elucidate their expression in a variety of conditions using S1-nuclease mapping techniques. However, in order to establish cause-effect relationships this descriptive approach is not sufficient. For this reason, we will use molecular genetic techniques in order to determine the role of putative regulatory sequences in the expression of the genes under study. The MHC and Alpha-TM genes already isolated will be mutagenized in vitro to generate well characterized and manipulable mini-genes. These in vitro modified genes will then be introduced into cardiac myocytes in culture as well as the mouse germ cell line and their expression during development and in response to a variety of stimuli studied. Further modification of these constructs should provide a detailed analysis of the sequences involved in the transcriptional regulation of cardiac MHC and the post-transcriptional processes responsible for the generation of several different Alpha-TM proteins from a single gene. Therefore, the proposal is divided into three main areas of research: a) Production and characterization of a cardiogenic cell line; b) Further characterization and in vivo analysis of the MHC genes, and c) Molecular analysis of the Alpha-TM gene.